The focus of this research project is the elucidation of the pathways by which iron is assimilated, transported, stored, and utilized by the develoing erythrocyte. The cell model being used is the rabbit reticulocyte. In the past we have emphasized a study of methodologies used in reticulocyte studies and the mobilization of iron from specifically labeled 59Fe-ghosts by unlabeled lysate. The kinetics of iron moblization and fate of the iron in this system have been examined in detail. The proposed work will involve three aspects of this system. First, we will complete a study of the kinetics and fate of iron mobilized by reticulocyte lysate from specifically labeled 59Fe-ghosts which are annealed to Biogel P-2 polyacrylamide beads using the method of Jacobson and Brandon (B.S. Jacobson and D. Brandon, 1977, Science 195, 302-304.). The advantage of this technique is that the membranes are presented to the lysate in an "inside-out" configuration. The second aspect of the proposed research is the completion of a study of the ability of specifically labeled 59Fe-ferritin to donate iron for hemoglobin synthesis in rabbit reticulocytes and bone marrow cells. Our results, to date, using the reticulocyte lysate indicate no donation of iron from ferritin. This is in conflict with literature reports. The third aspect which will begin shortly is a study of the possible role of metallothionein as an intracellular iron transport agent. It is quite likely that metallothionein is able to bind ferrous ion. This protein will be isolated from the kidney and spleen of rabbits treated with cadmium, since this should provide much larger amounts of the protein than is possible to obtain from reticulocytes. The ability of metallothionein to mobilize iron from 59Fe-ghosts and to deliver iron for hemoglobin and ferritin biosynthesis will be examined. Chemical aspects of iron binding by metallothionein will also be examined.